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Paper has been used to engineer many types of bio-diagnostics. A major issue to most paper-based bio-diagnostics is the biomolecule instability causing the short shelf-life of the diagnostics. Commercial papers contain various polymeric additives. Polyamidoamine-epichlorohydrin (PAE), a polyelectrolyte typically used as wet-strength agent, is commonly used in filter papers and paper towels, which are often used as substrate in bioactive paper. However, the effect of cellulose or polymeric additives on antibody bioactivity is unknown. This limits paper optimization for diagnostic applications. In this study, model papers were made with and without PAE addition. IgM Anti-A blood typing antibody was physisorbed and dried on paper, aged for up to 9 weeks at different relative humidity (RH) conditions and the antibody activity was measured. The antibody bioactivity was represented as blood typing efficiency measured by image analysis. The surface chemical composition was measured using X-ray photoelectron spectroscopy (XPS). Antibody bioactivity loss was promoted by elevated RH, corresponding to increased paper water content. PAE significantly reduces the paper water content under ambient environment. Antibody bioactivity is higher on paper made with PAE under the high humidity conditions (57.6%–84.3% RH). However, under conditions of humidity saturation (100%RH), PAE shows little effect on reducing paper water content nor on protecting antibody bioactivity. These results demonstrate the water content of paper to be associated with antibody bioactivity loss. [ABSTRACT FROM AUTHOR]